DESCRIPTION (Abstract of the application) The objective of this research is to develop novel retroviral vectors to treat diabetic rats with the long-term goal of treating patients with insulin dependent diabetes. We hypothesize that retroviral vectors encoding glucose responsive promoters driving furin expression may provide an amplified secretion of mature insulin. To achieve this we will construct novel 3-gene retroviral vectors with an insulin cleaving protease under the control of a glucose responsive promoter. We propose that protease expression will catalyze an amplified release of mature bioactive insulin in response to increased glucose concentration. Protease-mediated mature insulin release will be beyond that achieved by control of insulin alone. The vectors will be used to transduce vascular smooth muscle cells which are easily transduced with retroviral vectors, can be selected and returned to donor animals and express therapeutic genes for at least 1 year. The hypothesis that constitutive expression of secretable GAD65 will tolerize diabetic rats and prevent or significantly delay the onset of diabetes will be tested. In this aim we will use retroviral vectors containing elements of a fibronectin promoter to achieve long-term secretable GAD65 expression in transduced skin fibroblasts introduced into rats as skin equivalent grafts. In a third aim we will attempt to achieve in vivo retroviral infection/transduction of skin epidermal cells and will monitor a marker gene and vectors expressing secretable GAD65. The proposed vectors will be tested in a well characterized rat model of insulin dependent diabetes. The presence of homozygous lyp/lyp results in insulin-dependent diabetes in 100% of rats. The control litter mates, lyp/+ and +/+ are diabetes resistant. The onset of diabetes in the lyp/lyp rats is at 60-90 days of age. This well characterized diabetic BB rat therefore permits a unique model to study the efficacy of our novel vectors to a) control diabetes by hyperglycemia regulatable release of mature insulin and b) induce immune tolerance by secretion of the major autoantigen in diabetes, GAD65. These studies will test, in a well characterized rat model of diabetes, the ability to achieve a regulated supply of insulin at a clinically meaningful level, and to determine if tolerization can be achieved by secretion of an antigen involved in the pathogenesis of autoimmune diabetes. These studies of diabetic rats provide a model of regulated hormone gene therapy and have a potential application to other diseases requiring long-term delivery of therapeutic proteins such as cytokines, hormones, clotting factors and enzymes.